Cytokines such as interleukin-12 (IL-12) mediate the acute phase response to inflammatory stimuli, enhance the microbicidal functions of macrophages and other cells, and promote specific lymphocyte responses. See, e.g., Fearon and Locksley, Science 272:50 (1996).
Recently, in vivo studies revealed that inhibition of IL-12 production has therapeutic effects against inflammatory disorders such as sepsis (Zisman et al., Eur. J. Immunol. 27:2994 (1997)), collagen induced arthritis (Malfait et al., Clin. Exp. Immunol. 111:377 (1998)), established colitis (U.S. Pat. No. 5,853,697), experimental autoimmune encephalomyelitis (Leonard et al., J. Exp. Med. 181:381 (1995)), experimental autoimmune uveoretinitis (Yokoi et al., Eur. J. Immunol. 27:641 (1997)), psoriasis (Turka et al., Mol. Med. 1:690 (1995)), and cyclophosphamide induced diabetes (Rothe et al., Diabetologia 40:641 (1997)).
Production of IL-12 can be inhibited by anti-IL-12 antibodies. However, long term treatments of chronic diseases with such antibodies are expensive. Also, antibodies can be unstable after administration. Thus, there exists a need for use of small non-protein compounds instead of anti-IL-12 antibodies to inhibit the production of IL-12.
An aspect of this invention relates to a compound of formula (I): 
X is triazinyl. L1 is xe2x80x94A1xe2x80x94B1xe2x80x94, in which xe2x80x94A1xe2x80x94 is xe2x80x94(CH(Ra))mxe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, or xe2x80x94N(Rb)xe2x80x94 and xe2x80x94B1xe2x80x94 is xe2x80x94(CH(Rc))nxe2x80x94 or a bond. Each of Ra and Rc, independently, is hydrogen, alkyl, alkoxy, hydroxyl, hydroxylalkyl, carboxyl, halo, haloalkyl, amino, aminoalkyl, thio, thioalkyl, cyano, nitro, alkylcarbonylamino, alkylaminocarbonyl, formyl, alkylcarbonyl, alkylcarbonylalkyl, alkoxycarbonyl alkylcarbonyloxy, cycloalkyl, heterocycloalkyl, aryl, aralkyl, heteroaryl, or heteroaralkyl. Rb is hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aryl, aralkyl, heteroaryl, or heteroaralkyl. Each of m and n, independently, is 1-8. W is cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which being optionally substituted with alkyl, alkoxy, hydroxyl, hydroxylalkyl, carboxyl, halo, haloalkyl, amino, aminoalkyl, thio, thioalkyl, cyano, nitro, alkylcarbonylamino, alkylaminocarbonyl, formyl, alkylcarbonyl, alkylcarbonylalkyl, alkoxycarbonyl, or alkylcarbonyloxy. L2 is xe2x80x94A2xe2x80x94B2xe2x80x94, in which A2 is a bond, xe2x80x94N(R1)xe2x80x94, or xe2x80x94(xe2x80x94C(R2)(R3)xe2x80x94)pxe2x80x94, and B2 is a bond, xe2x80x94Nxe2x95x90C(R4)xe2x80x94, xe2x80x94C(R5)xe2x95x90Nxe2x80x94, xe2x80x94C(R6)xe2x95x90C(R7)xe2x80x94, xe2x80x94N(R8)xe2x95x90N(R9)xe2x80x94, xe2x80x94N(R10)xe2x80x94C(R11)(R12)xe2x80x94, xe2x80x94Oxe2x80x94C(R13)(R14)xe2x80x94, xe2x80x94COxe2x80x94C(R15)(R16)xe2x80x94, xe2x80x94COxe2x80x94N(R17)xe2x80x94, xe2x80x94N(R18)xe2x80x94COxe2x80x94, xe2x80x94COxe2x80x94, xe2x80x94COxe2x80x94Oxe2x80x94, xe2x80x94COxe2x80x94Sxe2x80x94, xe2x80x94Sxe2x80x94C(R19)(R20)xe2x80x94, xe2x80x94CSxe2x80x94C(R21)(R22)xe2x80x94, xe2x80x94CSxe2x80x94N(R23)xe2x80x94, xe2x80x94N(R24)xe2x80x94CSxe2x80x94, xe2x80x94CSxe2x80x94, xe2x80x94SO2xe2x80x94, provided that xe2x80x94A2xe2x80x94B2xe2x80x94 cannot be a bond. xe2x80x94A2xe2x80x94B2xe2x80x94 together is xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94(xe2x80x94Oxe2x80x94(CH2)qxe2x80x94Oxe2x80x94)rxe2x80x94, xe2x80x94(xe2x80x94N(R25)xe2x80x94(CH2)8xe2x80x94COxe2x80x94)txe2x80x94, or xe2x80x94(xe2x80x94N(R26)xe2x80x94(CH2)uxe2x80x94N(R27)xe2x80x94)vxe2x80x94. Each of R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15, R16, R17, R18, R19, R20, R21, R22, R23, R24, R25, R26, and R27, independently, is hydrogen, alkyl, alkoxy, hydroxyl, hydroxyalkyl, halo, haloalkyl, amino, aminoalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl. Each of p, q, r, s, t, u, and v, independently, is 1, 2, or 3. Y is xe2x80x94Rxe2x80x2xe2x80x94Lxe2x80x2xe2x80x94Rxe2x80x3 wherein Rxe2x80x2 is a bond, or cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl, each of which optionally being substituted with alkyl, alkoxy, hydroxyl, hydroxylalkyl, carboxyl, halo, haloalkyl, amino, aminoalkyl, thio, thioalkyl, cyano, nitro, alkylcarbonylamino, alkylaminocarbonyl, formyl, alkylcarbonyl, alkylcarbonylalkyl, alkoxycarbonyl, alkylcarbonyloxy, or alkoxycarbonylimino. Lxe2x80x2 is a bond, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94N(R28)xe2x80x94, xe2x80x94N(R29)xe2x80x94COxe2x80x94, xe2x80x94COxe2x80x94N(R30)xe2x80x94, xe2x80x94COxe2x80x94Oxe2x80x94, or xe2x80x94Oxe2x80x94COxe2x80x94. Each of R28, R29, and R30, independently, is hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl. Rxe2x80x3 is cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl, each of which being optionally substituted with alkyl, alkoxy, hydroxyl, hydroxyalkyl, carboxyl, halo, haloalkyl, amino, aminoalkyl, thio, thioalkyl, cyano, nitro, alkylcarbonylamino, alkylaminocarbonyl, formyl, alkylcarbonyl, alkylcarbonylalkyl, alkoxycarbonyl, or alkylcarbonyloxy. Z is morpholinyl which is optionally substituted with alkyl, alkoxy, hydroxyl, hydroxylalkyl, carboxyl, halo, haloalkyl, amino, aminoalkyl, thio, thioalkyl, cyano, nitro, alkylcarbonylamino, alkylaminocarbonyl, formyl, alkylcarbonyl, alkylcarbonylalkyl, alkoxycarbonyl, or alkylcarbonyloxy.
Another aspect of this invention relates to a method of inhibiting IL-12 production, which includes administering to a patient in need thereof an effective amount of a compound of formula (I), supra. X is triazinyl. L1 is xe2x80x94A1xe2x80x94B1xe2x80x94, in which xe2x80x94A1xe2x80x94 is xe2x80x94(CH(Ra))mxe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, or xe2x80x94N(Rb)xe2x80x94 and xe2x80x94B1 xe2x80x94 is xe2x80x94(CH(Rc))nxe2x80x94 or a bond. Each of Ra and Rc, independently, is hydrogen, alkyl, alkoxy, hydroxyl, hydroxylalkyl, carboxyl, halo, haloalkyl, amino, aminoalkyl, thio, thioalkyl, cyano, nitro, alkylcarbonylamino, alkylaminocarbonyl, formyl, alkylcarbonyl, alkylcarbonylalkyl, alkoxycarbonyl, alkylcarbonyloxy, cycloalkyl, heterocycloalkyl, aryl, aralkyl, heteroaryl, or heteroaralkyl, Rb is hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aryl, aralkyl, heteroaryl, or heteroaralkyl. Each of m and n, independently, is 1-8. W is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, or heteroaryl, each of which being optionally substituted with alkyl, alkoxy, hydroxyl, hydroxylalkyl, carboxyl, halo, haloalkyl, amino, aminoalkyl, thio, thioalkyl, cyano, nitro, alkylcarbonylamino, alkylaminocarbonyl, formyl, alkylcarbonyl, alkylcarbonylalkyl, alkoxycarbonyl, or alkylcarbonyloxy. L2 is xe2x80x94A2xe2x80x94B2xe2x80x94, in which A2 is a bond, xe2x80x94N(R1)xe2x80x94, or xe2x80x94(xe2x80x94C(R2)(R3)xe2x80x94)pxe2x80x94, and B2 is a bond, xe2x80x94Nxe2x95x90C(R4)xe2x80x94, xe2x80x94C(R5)xe2x95x90Nxe2x80x94, xe2x80x94C(R6)xe2x95x90C(R7)xe2x80x94, xe2x80x94N(R8)xe2x95x90N(R9)xe2x80x94, xe2x80x94N(R10)xe2x80x94C(R11)(R12)xe2x80x94, xe2x80x94Oxe2x80x94C(R13)(R14)xe2x80x94, xe2x80x94COxe2x80x94C(R15)(R16)xe2x80x94, xe2x80x94CO-xe2x80x94N(R17)xe2x80x94, xe2x80x94N(R18)xe2x80x94COxe2x80x94, xe2x80x94COxe2x80x94, xe2x80x94COxe2x80x94Oxe2x80x94, xe2x80x94COxe2x80x94Sxe2x80x94, xe2x80x94Sxe2x80x94C(R19)(R20)xe2x80x94, xe2x80x94CSxe2x80x94C(R21)(R22)xe2x80x94, xe2x80x94CSxe2x80x94N(R23)xe2x80x94, xe2x80x94N(R24)xe2x80x94CSxe2x80x94, xe2x80x94CSxe2x80x94, xe2x80x94SO2xe2x80x94, provided that xe2x80x94A2xe2x80x94B2xe2x80x94 cannot be a bond. xe2x80x94A2xe2x80x94B2xe2x80x94 together is xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94(xe2x80x94Oxe2x80x94(CH2)qxe2x80x94Oxe2x80x94)rxe2x80x94, xe2x80x94(xe2x80x94N(R25)xe2x80x94(CH2)sxe2x80x94COxe2x80x94)txe2x80x94, or xe2x80x94(xe2x80x94N(R26)xe2x80x94(CH2)uxe2x80x94N(R27)xe2x80x94)vxe2x80x94. Each of R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15,R16, R17, R18, R19, R20, r21, R22, R23, R24, R25, R26, and R27, independently, is hydrogen, alkyl, alkoxy, hydroxyl, hydroxylalkyl, halo, haloalkyl, amino, aminoalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl. Each of p, q, r, s, t, u, and v, independently, is 1, 2, or 3. Y is xe2x80x94Rxe2x80x2xe2x80x94Lxe2x80x2xe2x80x94Rxe2x80x3 wherein Rxe2x80x2 is a bond, or cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl, optionally substituted with alkyl, alkoxy, hydroxyl, hydroxylalkyl, carboxyl, halo, haloalkyl, amino, aminoalkyl, thio, thioalkyl, cyano, nitro, alkylcarbonylamino, alkylaminocarbonyl, formyl, alkylcarbonyl, alkylcarbonylalkyl, alkoxycarbonyl, alkylcarbonyloxy, or alkoxycarbonylimino. Lxe2x80x2 is a bond, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94N(R28)xe2x80x94, xe2x80x94N(R29)xe2x80x94COxe2x80x94, xe2x80x94COxe2x80x94N(R30)xe2x80x94, xe2x80x94COxe2x80x94Oxe2x80x94, or xe2x80x94Oxe2x80x94COxe2x80x94. Each of R28, R29, and R30, independently, is hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl. Rxe2x80x3 is cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl, each of which optionally being substituted with alkyl, alkoxy, hydroxyl, hydroxylalkyl, carboxyl, halo, haloalkyl, amino, aminoalkyl, thio, thioalkyl, cyano, nitro, alkylcarbonylamino, alkylaminocarbonyl, formyl, alkylcarbonyl, alkylcarbonylalkyl, alkoxycarbonyl, or alkylcarbonyloxy. Z is cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which being optionally substituted with alkyl, alkoxy, hydroxyl, hydroxylalkyl, carboxyl, halo, haloalkyl, amino, aminoalkyl, thio, thioalkyl, cyano, nitro, alkylcarbonylamino, alkylaminocarbonyl, formyl, alkylcarbonyl, alkylcarbonylalkyl, arkoxycarbonyl, or alkylcarbonyloxy.
Set forth below are exemplary of compounds of this invention: 
A pharmaceutically acceptable salt of a compound of formula (I) is also within the scope of this invention. For example, a salt can be formed between a negatively charged substituent such as carboxylate and a positively charged counterion such as an alkali metal ion (e.g., a sodium ion or a potassium ion); an alkaline earth metal ion (e.g., a magnesium ion or a calcium ion); an ammonium ion (NH4+); or an organic ammonium group such as tetramethylammonium ion or diisopropylethyl-ammonium ion. As another example , if an amino substituent can form a salt with a negatively charged counterion. Suitable counterions include, but are not limited to, chloride, hydrochloride, bromide, iodide, sulfate, nitrate, phosphate, or acetate.
As used herein, alkyl is a straight or branched hydrocarbon chain containing 1 to 12 carbon atoms. Examples of alkyl include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylhexyl, 3-ethyloctyl, and 4-ethyldecyl.
The term xe2x80x9calkenylxe2x80x9d refers to a straight or branched hydrocarbon chain containing 2 to 12 carbon atoms and one or more (e.g., 1-6) double bonds. The term xe2x80x9calkynylxe2x80x9d also refers to such a hydrocarbon chain, except that the chain contains one or more triple bonds instead of double bonds. Some examples of alkenyl and alkynyl are allyl, 2-butenyl, 2-pentenyl, 2-hexenyl, 2-butynyl, 2-pentynyl and 2-hexynyl.
By cycloalkyl is meant a cyclic alkyl group containing 3 to 8 carbon atoms. Some examples of cycloalkyl are cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, and norbornyl. Heterocycloalkyl is a cycloalkyl group containing 1-3 heteroatoms such as nitrogen, oxygen, or sulfur. Examples of heterocycloalkyl include piperidinyl, piperazinyl, tetrahydropyranyl, tetrahydrofuryl, and morpholinyl. Cycloalkenyl is a cycloalkyl group containing one or more (e.g., 1-3) double bonds. Examples of such a group include cyclopentenyl, 1,4-cyclohexadienyl, cycloheptenyl, and cyclooctenyl groups. By the same token, heterocycloalkenyl is a heterocycloalkyl group containing one or more double bonds.
As used herein, aryl is an aromatic group containing 6-12 ring atoms and can contain fused rings, which may be saturated, unsaturated, or aromatic, Examples of an aryl group include phenyl, naphthyl, biphenyl, phenanthryl, and anthracyl. Heteroaryl is aryl containing 1-3 heteroatoms such as nitrogen, oxygen, or sulfur and can contain fused rings. Some examples of heteroaryl are pyridyl, furanyl, pyrrolyl, thienyl, thiazolyl, oxazolyl, imidazolyl, indolyl, benzofuranyl, and benzthiazolyl.
A cyclic moiety can be cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, or heteroaryl. A tricyclic moiety is a fused ring formed from three of the just-mentioned moieties. An example of a tricyclic moiety is dibenzocycloheptenyl. Heteroatoms such as nitrogen, oxygen, and sulfur can be included in the cyclic moiety.
An amino group can be unsubstituted, mono-substituted, or di-substituted. It can be substituted with groups such as alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl. Halo refers to fluoro, chloro, bromo, or iodo.
Compounds of this invention show an unexpected high level of potency in inhibiting the production of IL-12.
Other features or advantages of the present invention will be apparent from the following detailed description of several embodiments, and also from the appending claims.
A compound of formula (I) can be prepared in a stepwise manner by using cyanuric chloride as a starting material. Cyanuric chloride is commercially available. The three chloro groups of cyanuric chloride can be successively displaced by various nucleophilic substituents. The order of displacement is not of particular importance. For example, a chloro group of cyanuric chloride can be substituted with a nucleophile Wxe2x80x94B1xe2x80x94A1xe2x80x94H wherein A1 is xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, or xe2x80x94N(Rb)xe2x80x94, and W and B1 have been defined above, thus forming an ether linkage. Below is an example of a displacement reaction in which the nucleophile is phenethyl alcohol (i.e., A1 is xe2x80x94Oxe2x80x94, B1 is xe2x80x94CH2xe2x80x94CH2xe2x80x94, and W is phenyl): 
A compound of formula (I) wherein A1 is xe2x80x94(CH(Ra))mxe2x80x94 (Ra and m have been defined above) can be prepared by first converting cyanuric chloride to an organometallic compound of group IA or IIA metal, e.g., a Grignard reagent. The organometallic compound acts as a nucleophile and reacts with a compound with a good leaving group, e.g., an alkyl halide, to form a carbon-carbon linkage.
A chloro group of cyanuric chloride can also be displaced by a nucleophile ZH (Z has been defined above), e.g., morpholine, to form a morpholinyl triazine compound as shown in the following reaction (see, e.g., Step (2) of Example 1 for details): 
A chloro group of cyanuric chloride can undergo yet another displacement reaction with a nucleophile Hxe2x80x94A2xe2x80x94B2xe2x80x94Y wherein A2 is an amine and B2 is a bond to form an amino linkage between the triazine ring and the nucleophile. Some examples of such an amine include 3-aminocarbazole, 6-aminobenzofuran, and 4-(3-methylphenyl)aniline. See, e.g., Step (3) of Example 5. On the other hand, as shown in the following reaction, if the desired linkage xe2x80x94A2xe2x80x94B2xe2x80x94 is xe2x80x94NHxe2x80x94Nxe2x95x90CHxe2x80x94, the chloro group can be first displaced by hydrazine, and the primary amine of the coupled hydrazine moiety can then react with an aldehyde to form an imine linkage: 
A compound of formula (I) wherein the linkage xe2x80x94A2xe2x80x94B2xe2x80x94 is a carbon-carbon linkage can be formed, e.g., via an organometallic intermediate described above and further reacting such an intermediate with a nucleophile.
Other types of linkages can be prepared by similar nucleophilic reactions. Sensitive moieties on both the triazinyl intermediates and the nucleophiles can be protected prior to coupling. For suitable protecting groups, see, e.g., Greene, T. W., Protective Groups in Organic Synthesis, John Wiley and Sons, Inc., New York (1981).
A compound of formula (I) prepared by the methods shown above can be purified by flash column chromatography, preparative high performance liquid chromatography, or crystallization.
A pharmaceutical composition containing an effective amount of one or more compounds of this invention for inhibiting IL-12 production or treating an IL-12 mediated disorders is also within the scope of this invention. Some examples of IL-12 mediated disorders include sepsis and autoimmune disorders, e.g., rheumatoid arthritis, Crohn""s disease, psorasis, and multiple sclerosis. The use of a compound of this invention or a salt thereof for the manufacture of a medicament for inhibiting IL-12 production or treating the above-mentioned IL-12-mediated disorders is also within the scope of this invention. Still another aspect of this invention is a method of treating an IL-12-mediated disorder, e.g., sepsis, by administering to a patient a pharmaceutical composition containing an effective amount of a compound of this invention or a salt thereof. An effective amount is defined as the amount which is required to confer a therapeutic effect on the treated patient, and is typically determined based on age, surface area, weight, and condition of the patient. The interrelationship of dosages for animals and humans (based on milligrams per meter squared of body surface) is described by Freireich et al., Cancer Chemother. Rep. 1966, 50, 219. Body surface area may be approximately determined from height and weight of the patient. See, e.g., Scientific Tables, Geigy Pharmaceuticals, Ardley, N.Y., 1970, 537. An effective amount of a compound of this invention can range from about 0.1 mg/kg to about 100 mg/kg. Effective doses will also vary, as recognized by those skilled in the art, dependant on route of administration, excipient usage, and the possibility of co-usage with other therapeutic treatments such as the use of other anti-inflammatory agents.
The pharmaceutical composition may be administered via the parenteral route, including orally, topically, subcutaneously, intraperitoneally, intramuscularly, and intravenously. Examples of parenteral dosage forms include aqueous solutions, isotonic saline or 5% glucose of the active agent, or other well-known pharmaceutically acceptable excipient. Solubilizing agents such as cyclodextrins, or other solubilizing agents well-known to those familiar with the art, can be utilized as pharmaceutical excipients for delivery of the therapeutic compounds.
A compound of this invention can be formulated into dosage forms for other routes of administration utilizing conventional methods. For example, it can be formulated in a capsule, a gel seal, or a tablet for oral administration. Capsules may contain any standard pharmaceutically acceptable materials such as gelatin or cellulose. Tablets may be formulated in accordance with conventional procedures by compressing mixtures of a compound of this invention with a solid carrier and a lubricant. Examples of solid carriers include starch and sugar bentonite. A compound of this invention can also be administered in a form of a hard shell tablet or a capsule containing a binder, e.g., lactose or mannitol, a conventional filler, and a tableting agent.
The biological activities of a compound of this invention can be evaluated by a number of cell-based assays. One of such assays can be conducted using mononuclear cells from human peripheral blood (PBMC). Human Interferon gamma (IFN-xcex3) and lipopolysaccharide are added to the cell-containing solutions to induce IL-12 production. A solution containing a compound of this invention is added to each well. The level of inhibition of IL-12 production can be measured by determining the amount of P70 (i.e., IL-12) by using a sandwich ELISA assay with anti-human IL-12 antibodies. IC50 of each test compound can then be determined and potent compounds are selected for further testing.
The selected compounds can be further assayed in parallel on two types of cells, PBMC and human leukemia mononuclear cell line (THP-1), for verification. More specifically, each test compound is added to a well of stimulated PBMC or THF-1 cells. PBMC are stimulated by a combination of IFN-xcex3 and pansorbin. The amount of P70 present in each well is then measured using a sandwich ELISA with anti-human IL-12 antibodies and IC50 is determined for each test compound.
The cytotoxity of the test compounds of this invention can be determined by using a bioreductive assay. Specifically, PBMC and THP-1 cells are incubated with the test compounds. Viability are evaluated by the ability of mitochondrial dehydrogenases of the incubated cells to reduce 3-(4,5-dimethylthiazole-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) in the presence of phenazine methosulfate (PMS).
Compounds of this invention can also be evaluated by animal studies. One of such studies involves the ability of a test compound to inhibit septic shock. Animals are injected with LPS to induce septic shock. Mortality rate of the induced animals is monitored after administering to the animals a test compound and compared with that of a control experiment in which the induced animals are not treated.
The following specific examples, which describe syntheses and biological testings of compounds of this invention, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
In the following examples, 1H nuclear magnetic resonance spectra were recorded on a Varian Mercury 300 MHz spectrometer. ES mass spectra were recorded on a Finnigan Navigator mass spectrometer.
Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. The following specific examples, which described syntheses, screening, and biological testing of various compounds of this invention, are therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications recited herein, including patents, are hereby incorporated by reference in their entirety.